The broad long-term objective of this proposal is to understand both the biochemical mechanism and biological function of DNA helicase action, i.e., how helicases translocate along single-stranded DNA (ssDNA) to unwind double-stranded DNA (dsDNA) and, how this effort is translated into biological function. Furthermore, one of the helicases (RecBCD) that is a major focus of this proposal recognizes a specific sequence (G) while translocating and, in response, alters its biochemical behavior with unprecedented complexity. The second helicase (RecQ) is seemingly less complex, but it works in conjunction with a topoisomerase (topoisomerase III), to change DNA topology in a novel way. Understanding the mechanism and function of these motor proteins is a longstanding goal of this research proposal. This grant proposal has 3 broad specific aims. The first is to extend our single-molecule analysis of RecBCD helicase so that we can visualize the binding of RecA protein to RecBCD enzyme, as well as the loading of RecA protein onto G-containing ssDNA. The second is to use the recent crystal structure to guide functional analysis of RecBCD enzyme to understand the mechanism of translocation and the consequences of G-recognition. The third is to characterize RecQ helicase and its interaction with topoisomerase III by visualizing translocation RecQ helicase at the single-molecule level behavior, and studying Holliday junction migration by the concerted actions of these two proteins. Understanding how these motor proteins accomplish these tasks is the overall goal of this proposal. To accomplish this goal, biochemical, enzymatic, structural- functional, and single-molecule analyses are planned. This research will provide basic information about protein-DNA interactions;insight into the behavior of molecular motors, their translocation process, and their application as nanomachines;and an appreciation of the molecular events underlying cellular aberrations that cause disease. Mutations in genes that encode putative helicases are associated with human pathologies, such as breast cancer and Fanconi's anemia (BACH1/BRIP1/FANCJ;xeroderma pigmentosum (ERCC2 and ERCC3);Cockayne's (ERCC6);Bloom's (BLM);Werner's (WRN);and Rothmund-Thomson (RECQ4), showing that an understanding of these proteins is crucial for the understanding of disease processes as diverse as cancer, anemia, and premature aging. Project Narrative: The broad objective of this proposal is to understand both the biochemical mechanism and biological function of DNA helicases. These proteins are involved in various aspects of DNA repair and chromosome maintenance. Mutations in related proteins are associated with human diseases as diverse as cancer, anemia, and aging.